This proposal takes advantage of a unique natural experiment in ion channel structure and function that produced variants of a TRPN channel, encoded in Drosophila by the no mechanoreceptor potential C (nompC) gene. TRPN channels are sensory mechanotransducers, found both in invertebrate mechanosensory neurons and in some vertebrate hair cells. Sensory mechanotransduction is the conversion of mechanical stimuli to cellular potentials: it underlies our senses of touch, hearing, balance and proprioception. Previous work showed that NOMPC is needed to transduce touch and sound, and is located at the tips of sensory cilia in several different mechanoreceptor organs. We have now described multiple NOMPC isoforms, produced by alternate transcript splicing. Most unusually for any channel protein, they encode different versions of the probable pore-forming region, which sets channel conductance and ion selectivity. Moreover, similar pore isoforms are found in other insect orders - notably, those that have evolved high-speed, maneuverable flight. We hypothesize that they reflect the diversification of insect mechanoreceptor organs, possibly under selection from the sensory demands of high-speed flight, and form channels that are differently optimized for specific receptor organs. To test this, we will make transgenic fly strains that each express only one NOMPC isoform, by expressing cDNA constructs in a nompC mutant background; isoforms can be combined by crossing the transgenic strains together. In a complementary approach, RNA interference directed against each alternate exon will selectively downregulate specific isoforms. The ability of each isoform or combination to function in the different sense organs will be tested by behavioral assays and by electrophysiological recording. Finally, the natural splice pattern will be made visible at single-neuron resolution by transgenic splice-reporter genomic constructs, fluorescently tagged in alternate exons. The results will give insights into the structural determinants of mechanotransducer channel activation and adaptation, the operation of TRP superfamily channels in general, and the evolution of a mechanosensory system at the molecular, physiological and morphological levels. PUBLIC HEALTH RELEVANCE: The medical significance of the proposal lies in its potential insights into mechanotransduction and into the operation of TRP superfamily channels. Although NOMPC was one of the first identified mechanosensory channels, the mechanism by which it or any eukaryotic mechanosensory channel is activated and regulated is still unknown. The TRP superfamily, to which TRPN channels belong, includes cation channels that transduce many sensory stimuli and physiological signals, including noxious heat and pain, as well as mechanical signals. They are associated with a growing list of diseases and pathologies, including night blindness, several forms of kidney disease, neurodegeneration (Charcot-Marie-Tooth disease), and gastrointestinal and neuropathic pain. Many TRP channels are expressed on cilia, which themselves are increasingly recognized as centers or developmental and sensory signal transduction and integration. Despite their functional diversity, sequence conservation close to the pore region suggests a fundamentally similar conformation and gating mode for transmembrane regions of the different types. Thus, findings from the TRPN channel variants, and their role in insect mechanosensory cilia may be very broadly applicable.